The goal of this research program is to determine how gene expression is regulated in mammalian cells. One of the key steps in the pathway of messenger RNA production may involve small nuclear RNA (snRNA) molecules. We have shown by chemical cross-linking with psoralen that some of these small RNA species actually interact with precursors of messenger RNA and ribosomal RNA. We plan now to show how these interactions occur, and in addition, to determine how these snRNAs are synthesized, and whether their own biosynthesis is coupled to that of messenger (and ribosomal) RNA. This will be accomplished by continuing to explore RNA-RNA base-pairing interactions in vivo using psoralen cross-linking to determine which snRNAs associate with high molecular weight nuclear precursors of messenger RNA and ribosomal RNA. Recombinant clones bearing snRNA genes will be utilized as highly sensitive and specific hybridization probes to detect cross-linked snRNAs. To analyze specific snRNA:mRNA precursor interactions using a combination of psoralen cross-linking and cloned snRNA DNA sequences, S1 nuclease mapping and sequencing techniques will be used to map the actual sites on the snRNAs and on the precursors that base-pair.